Cytochromes P-450 and P-448 are the membrane-bound terminal oxidases responsible for the metabolism of drugs and carcinogens, respectively, in mammalian liver. These cytochromes are believed to exist within the matrix of the smooth endoplasmic reticulum in several molecular forms with different chemical, physical and biological properties, which include substrate specificity, ligand binding equilibria and kinetics, molecular weight, spectral, and immunologic properties. The goal of this research is to assess the extent to which solubilization and purification of these cytochromes from the microsomal membrane change some of these properties. Specifically, the kinetics of CO binding and dissociation will be used as a model ligand for O2, as well as to probe subtle changes in the relationship between the prosthetic heme group and the apoprotein. An important feature of this work is that the purified cytochromes will be reincorporated singly and in various combinations into the hepatic microsomal vesicles obtained from untreated rats, which are themselves low in endogenous cytochrome P-450 and P-448. These reconstitution experiments are expected to indicate the reversibility (or lack of it) of any changes in the cytochromes brought about by the isolation procedures, and to indicate also the possibility that interactions between different types of cytochrome could occur. In addition, I propose to incorporate the purified cytochromes into synthetic phospholipid vesicles as a means of studying the importance of lipid-cytochrome interaction in CO binding and in mixed-function oxidase activity.